This project is concerned with the tumor-inducing pTiC58 plasmid of Agrobacterium tumefaciens. The pTiC58 plasmid contains the information required for virulence and oncogenicity. This information is contained in two regions of pTiC58. Work is focused on these regions and involves determining: (1)\the number of genes required for virulence and oncogenicity; (2)\the precise location of these genes; (3)\the mechanism by which these genes are regulated; and (4) the identification of the products of these genes. We are also constructing a protein map relative to the position of these genes by using a homologous coupled transcription-translation system, immunologic probes, and newly constructed amplifiable cloning plasmid cloning and expression vectors. Each of the genes of the Vir region so far studied are absolutely essential for virulence and oncogenicity. We have discovered that some of these genes are regulated by the inducer indole-3-acetic acid, while others are regulated by an unidentified substance which is elaborated by the plant during active cell division. The identification of inducers and the construction of gene fusions has allowed us for the first time to determine the timing and mechanism by which Vir genes are switched on during early stages of bacterial-plant interactions leading to tumor formation. We are extending similar efforts to the T-region, particularly on the regulation of genes that remain silent in the bacterium but are expressed in the eukaryotic plant cell. Studies are also being made on the precise location of the virulence suppressive genes contained in plasmid pSa and the products of these genes. Also, additional work is being done on characterizing plasmid pTAR and its origin of replication. (R)